increases the production of OspC shortly after an infected tick begins taking a blood meal (28
), and the expression of OspC is critical for establishing a mammalian infection (11
). OspC antibodies are therefore a hallmark of human Lyme disease (8
), and researchers have evaluated the ability of ELISAs based on OspC to provide serodiagnostic confirmation of the illness (19
). However, the ELISAs have lacked specificity because cross-reactive antibodies bind to heterogeneous regions of the protein (13
). The flow cytometric OspC borreliacidal antibody test circumvents this shortcoming (3
), but the necessity for viable spirochetes, the potential for interference from antimicrobial agents, and the technical complexity limit the usefulness. We therefore evaluated the ability of a synthetic-peptide ELISA based on the immunodominant epitope (18
) to provide serodiagnostic confirmation of early Lyme disease by detecting OspC borreliacidal antibodies.
The ELISA reactivities of sera from patients with other infections or autoimmune diseases were negligible, so the IgM and IgG tests could be adjusted to high specificity (98%) by using relatively low diagnostic OD cutoff values. At this level, false-positive IgM reactivity was detected for a single serum sample from a patient with CMV, and false-positive reactions were detected for three and five uncharacterized sera from patients undergoing cholesterol screening tests by the IgM and IgG ELISAs, respectively. The false-positive result from the CMV patient can be accounted for by the ability of the infection to cause polyclonal B-cell stimulation, but explanations for the reactivities in the other sera are less clear. It should be noted, however, that at least some reactivity may have been due to unrecognized Lyme disease. In support, the reactivities of sera from healthy blood donors who resided in a focus of nonendemicity (Milwaukee) were negligible. In addition, the Gundersen Medical Center is located in a focus of high endemicity (12
), and the identities and clinical histories of the patients being tested for cholesterol were unknown. Moreover, one of the IgM and IgG OspC7 ELISA-positive serum samples was also positive by the C6 ELISA (data not shown).
In contrast to the minimal reactivity detected in the potentially cross-reactive sera, the early Lyme disease sera reliably contained IgM OspC7-specific antibodies and some also contained IgG antibodies. Furthermore, the results from the IgM ELISA closely paralleled the findings from the flow cytometric borreliacidal-antibody test, and the ELISA was even more sensitive during the earliest stages of the illness. The latter finding is likely because of the increased efficiency of using purified “target” antigen rather than viable intact organisms. This result therefore corroborated previous reports (3
) that human borreliacidal antibodies detected by using B. burgdorferi
50772 are IgM or IgG OspC borreliacidal antibodies specific for the OspC C terminus and also confirmed that the ELISA could be used to detect the response.
In addition, the IgM OspC7 ELISA was significantly (P
< 0.001) more sensitive than an FDA-approved Western blotting procedure for confirming early Lyme disease. This is extremely significant, because the two-test system mandated currently by the CDC is cumbersome, time-consuming, and expensive. In response, alternative tests have been developed, and two in particular, the VlsE (2
) and C6 (17
) ELISAs, offer promise as stand-alone tests that confirm Lyme disease by detecting specific antibodies induced by multiple pathogenic Borrelia
). However, the tests primarily detect IgG antibodies (2
) and lack sensitivity during early Lyme disease (2
). In contrast, the OspC7 ELISA detects an immunodominant IgM borreliacidal antibody response produced early during infection. Therefore, the combination of high specificity and sensitivity provided by testing serum with both the IgM OspC7 ELISA and either the VlsE or C6 ELISA could conceivably eliminate the necessity for two-tiered testing.
Moreover, a BLAST search confirmed (18
) that the amino acid sequence recognized by the OspC7 antibodies is conserved among the pathogenic Borrelia
spp., despite the well-documented (13
) diversity of OspC alleles (13
) both within and among pathogenic Borrelia
genospecies. In addition, the OspC borreliacidal antibodies were detected by using a B. burgdorferi
sensu stricto isolate (50772) recovered from a host-seeking Ixodes scapularis
tick captured from a focus of endemicity in Connecticut (1
). Therefore, the OspC7 ELISA should be effective for confirming early Lyme disease in patients from throughout the United States and may also be useful for detecting the illness among European Lyme disease patients.
Interestingly, researchers (2
) previously evaluated an IgM ELISA based on a 10-amino-acid peptide (pepC10) that contained a sequence identical to that used in the OspC7 ELISA. The investigators confirmed that the ELISA detected specific antibodies in sera from patients infected with B. garinii
) or B. burgdorferi
), but the test appeared less sensitive than Western blotting when testing sera from patients with an EM lesion. We believe that there is a simple explanation for the discrepancy. The sera in our study were collected immediately prior to antibiotic therapy and stored frozen for short amounts of time without thawing. In contrast, many sera tested in the previous studies (2
) were stored and used for years, and IgM activity can be reduced significantly after one freeze-thaw cycle (26
). Furthermore, some sera were collected after the patient completed antibiotic therapy, and there is compelling evidence that the levels of borreliacidal antibodies, such as those detected by the pepC10 and OspC7 ELISAs, decrease rapidly after the antigen is eliminated. For example, Lyme disease vaccines that provide protection by inducing OspA borreliacidal antibodies to eliminate spirochetes from feeding ticks (9
) are effective for only a few months because the response disappears rapidly (24
In summary, additional studies to more completely define the utility of the OspC7 ELISA remain necessary. However, the findings confirm that the peptide based on the 7 C-terminal amino acids of OspC detects borreliacidal antibodies efficiently and provide strong evidence that the test can eliminate the necessity for two-tiered testing during early Lyme disease.